The Potential of Calcium Phosphate Nanoparticles as Adjuvants and Vaccine Delivery Vehicles

Vaccination is one of the most efficacious and cost-effective ways to protect people from infectious diseases and potentially cancer. The shift in vaccine design from disrupted whole pathogens to subunit antigens has brought attention on to vaccine delivery materials. For the last two decades, nanotechnology-based vaccines have attracted considerable attention as delivery vehicles and adjuvants to enhance immunogenicity, exemplified with the current COVID vaccines. The nanoparticle vaccines display unique features in protecting antigens from degradation, controlled antigen release and longer persisting immune response. Due to their size, shape and surface charge, they can be outstanding adjuvants to achieve various immunological effects. With the safety and biodegradable benefit of calcium phosphate nanoparticles (CaP NPs), they are an efficient carrier for vaccine design and adjuvants. Several research groups have studied CaP NPs in the field of vaccination with great advances. Although there are several reports on the overview of CaP NPs, they are limited to the application in biomedicine, drug delivery, bone regeneration and the methodologies of CaP NPs synthesis. Hence, we summarised the basic properties of CaP NPs and the recent vaccine development of CaP NPs in this review.

Enhancing Immune Responses to a DNA Vaccine Encoding Toxoplasma gondii GRA7 Using Calcium Phosphate Nanoparticles as an Adjuvant

Toxoplasma gondii infects almost all warm-blooded animals, including humans. DNA vaccines are an effective strategy against T. gondii infection, but these vaccines have often been poorly immunogenic due to the poor distribution of plasmids or degradation by lysosomes. It is necessary to evaluate the antigen delivery system for optimal vaccination strategy. Nanoparticles (NPs) have been shown to modulate and enhance the cellular humoral immune response. Here, we studied the immunological properties of calcium phosphate nanoparticles (CaPNs) as nanoadjuvants to enhance the protective effect of T. gondii dense granule protein (GRA7). BALB/c mice were injected three times and then challenged with T. gondii RH strain tachyzoites. Mice vaccinated with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) showed a strong cellular immune response, as monitored by elevated levels of anti-T. gondii-specific immunoglobulin G (IgG), a higher IgG2a-to-IgG1 ratio, elevated interleukin (IL)-12 and interferon (IFN)-γ production, and low IL-4 levels. We found that a significantly higher level of splenocyte proliferation was induced by GRA7-pEGFP-C2+nano-adjuvant (CaPNs) immunization, and a significantly prolonged survival time and decreased parasite burden were observed in vaccine-immunized mice. These data indicated that CaPN-based immunization with T. gondii GRA7 is a promising approach to improve vaccination.

Physiological and Molecular Investigation of Urea Uptake Dynamics in Cucumis sativus L. Plants Fertilized With Urea-Doped Amorphous Calcium Phosphate Nanoparticles

At present, the quest for innovative and sustainable fertilization approaches aiming to improve agricultural productivity represents one of the major challenges for research. In this context, nanoparticle-based fertilizers can indeed offer an interesting alternative with respect to traditional bulk fertilizers. Several pieces of evidence have already addressed the effectiveness of amorphous calcium phosphate-based nanoparticles as carriers for macronutrients, such as nitrogen (N), demonstrating increase in crop productivity and improvement in quality. Nevertheless, despite N being a fundamental nutrient for crop growth and productivity, very little research has been carried out to understand the physiological and molecular mechanisms underpinning N-based fertilizers supplied to plants via nanocarriers. For these reasons, this study aimed to investigate the responses of Cucumis sativus L. to amorphous calcium phosphate nanoparticles doped with urea (U-ACP). Urea uptake dynamics at root level have been investigated by monitoring both the urea acquisition rates and the modulation of urea transporter CsDUR3, whereas growth parameters, the accumulation of N in both root and shoots, and the general ionomic profile of both tissues have been determined to assess the potentiality of U-ACP as innovative fertilizers. The slow release of urea from nanoparticles and/or their chemical composition contributed to the upregulation of the urea uptake system for a longer period (up to 24 h after treatment) as compared to plants treated with bulk urea. This prolonged activation was mirrored by a higher accumulation of N in nanoparticle-treated plants (approximately threefold increase in the shoot of NP-treated plants compared to controls), even when the concentration of urea conveyed through nanoparticles was halved. In addition, besides impacting N nutrition, U-ACP also enhanced Ca and P concentration in cucumber tissues, thus having possible effects on plant growth and yield, and on the nutritional value of agricultural products.

MCP mediated active targeting calcium phosphate hybrid nanoparticles for the treatment of orthotopic drug-resistant colon cancer

Background Colon cancer is a most common malignant cancer in digestive system, and it is prone to develop resistance to the commonly used chemotherapy drugs, leading to local recurrence and metastasis. Paris saponin VII (PSVII) could not only inhibit the proliferation of colon cancer cells but also effectively induce apoptosis of drug-resistant colon cancer cells and reduce the metastasis of drug-resistant colon cancer cells as well. However, PSVII was insoluble in water and fat. It displayed no selective distribution in body and could cause severe hemolysis. Herein, colon cancer targeting calcium phosphate nanoparticles were developed to carry PSVII to treat drug-resistant colon cancer. Results PSVII carboxymethyl-β-cyclodextrin inclusion compound was successfully encapsulated in colon cancer targeting calcium phosphate nanoparticles (PSVII@MCP-CaP) by using modified citrus pectin as stabilizer agent and colon cancer cell targeting moiety. PSVII@MCP-CaP significantly reduced the hemolysis of PSVII. Moreover, by specific accumulating in orthotopic drug-resistant colon cancer tissue, PSVII@MCP-CaP markedly inhibited the growth of orthotopic drug-resistant colon cancer in nude mice. PSVII@MCP-CaP promoted the apoptosis of drug-resistant colon cancer cells through mitochondria-mediated apoptosis pathway. Moreover, PSVII@MCP-CaP significantly inhibited the invasion and migration of drug-resistant colon cancer cells by increasing E-cadherin protein expression and reducing N-cadherin and MMP-9 protein expression. Conclusion PSVII@MCP-CaP has great potential in the treatment of drug-resistant colon cancer. This study also explores a new method to prepare active targeting calcium phosphate nanoparticles loaded with a fat and water insoluble compound in water. Graphical Abstract

Incorporation of Functionalized Calcium Phosphate Nanoparticles in Living Cells

Intracellular calcium (Ca2+) is a key signaling element that is involved in a great variety of fundamental biological processes. Thus, Ca2+ deregulation would be involved in the cancer cell progression and damage of mitochondrial membrane and DNA, which lead to apoptosis and necrosis. In this study, we have prepared amorphous calcium phosphate nanoparticles (ACP NPs) for studied their incorporation by endocytosis or electroporation to epithelial, endothelial and fibroblast cells (MCF-7, HUVEC and COS-1 cells, respectively). Our results showed that internalized ACP NPs have cytotoxic effects as a consequence of the increase of the intracellular calcium content. The endocytosis pathways showed a greater cytotoxic effect since calcium ions could easily be released from the nanoparticles and be accumulated in the lysosomes and mitochondria. In addition, the cytotoxic effect could be reversed when calcium ion was chelated with ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Modification of ACP NPs by coating with different compounds based on phosphates was also evaluated. The results indicated a reduction of the cytotoxic effect, in the order polyphosphate < phosphonic acid < orthophosphate. A differential cytotoxic effect of ACP-NPs was observed in function of the cell type; the cytotoxic effect can be ordered as i.e., HUVEC > COS-1 > MCF-7. The greater cytotoxic effect caused by the increase of intracellular calcium that is observed in normal cells and the greater resistance of cancer cells suggests new perspectives for cancer research.

Inhalable Microparticles Embedding Calcium Phosphate Nanoparticles for Heart Targeting: The Formulation Experimental Design

Inhalation of Calcium Phosphate nanoparticles (CaPs) has recently unmasked the potential of this nanomedicine for a respiratory lung-to-heart drug delivery targeting the myocardial cells. In this work, we investigated the development of a novel highly respirable dry powder embedding crystalline CaPs. Mannitol was selected as water soluble matrix excipient for constructing respirable dry microparticles by spray drying technique. A Quality by Design approach was applied for understanding the effect of the feed composition and spraying feed rate on typical quality attributes of inhalation powders. The in vitro aerodynamic behaviour of powders was evaluated using a medium resistance device. The inner structure and morphology of generated microparticles were also studied. The 1:4 ratio of CaPs/mannitol led to the generation of hollow microparticles, with the best aerodynamic performance. After microparticle dissolution, the released nanoparticles kept their original size.